scholarly journals Multimeric structure of platelet factor VIII/von Willebrand factor: the presence of larger multimers and their reassociation with thrombin- stimulated platelets

Blood ◽  
1982 ◽  
Vol 60 (5) ◽  
pp. 1132-1138 ◽  
Author(s):  
MF Fernandez ◽  
MH Ginsberg ◽  
ZM Ruggeri ◽  
FJ Batlle ◽  
TS Zimmerman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Structural Difference ◽  
Platelet Factor ◽  
Cell Extracts ◽  
Platelet Enzymes ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The multimeric structure of platelet factor VIII/von Willebrand factor (FVIII/vWF) in cell extracts and in collagen and thrombin releasates has been analyzed by SDS polyacrylamide gel electrophoresis followed by detection with 125I-anti-FVIII/vWF. Platelets contained larger multimers than those normally present in plasma. When secreted FVIII/vWF was analyzed, all platelets. In contrast, in thrombin releasates the larger multimers were lost in a manner dependent on divalent cations, time, and thrombin dose. This loss could not be accounted for by modification of FVIII/vWF by thrombin or platelet enzymes since no effect of thrombin on the multimeric structure of FVIII/vWF in the absence of platelets or in the presence of platelet lysates was observed. Large multimers of 125I-labeled purified FVIII/vWF underwent divalent cation-dependent association with platelets in the presence of thrombin, indicating that the loss of FVIII/vWF from thrombin releasates was due to reassociation with the platelet. These studies show a structural difference between platelet and plasma FVIII/vWF that suggests a specific role for platelet FVIII/vWF in hemostasis.

scholarly journals Multimeric structure of platelet factor VIII/von Willebrand factor: the presence of larger multimers and their reassociation with thrombin- stimulated platelets

Blood ◽  
1982 ◽  
Vol 60 (5) ◽  
pp. 1132-1138 ◽  
Author(s):  
MF Fernandez ◽  
MH Ginsberg ◽  
ZM Ruggeri ◽  
FJ Batlle ◽  
TS Zimmerman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Structural Difference ◽  
Platelet Factor ◽  
Cell Extracts ◽  
Platelet Enzymes ◽  
Von Willebrand ◽  
Willebrand Factor

The multimeric structure of platelet factor VIII/von Willebrand factor (FVIII/vWF) in cell extracts and in collagen and thrombin releasates has been analyzed by SDS polyacrylamide gel electrophoresis followed by detection with 125I-anti-FVIII/vWF. Platelets contained larger multimers than those normally present in plasma. When secreted FVIII/vWF was analyzed, all platelets. In contrast, in thrombin releasates the larger multimers were lost in a manner dependent on divalent cations, time, and thrombin dose. This loss could not be accounted for by modification of FVIII/vWF by thrombin or platelet enzymes since no effect of thrombin on the multimeric structure of FVIII/vWF in the absence of platelets or in the presence of platelet lysates was observed. Large multimers of 125I-labeled purified FVIII/vWF underwent divalent cation-dependent association with platelets in the presence of thrombin, indicating that the loss of FVIII/vWF from thrombin releasates was due to reassociation with the platelet. These studies show a structural difference between platelet and plasma FVIII/vWF that suggests a specific role for platelet FVIII/vWF in hemostasis.

scholarly journals Factor VIII-von Willebrand factor requires calcium for facilitation of platelet adherence

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 996-103 ◽  
Author(s):  
KS Sakariassen ◽  
M Ottenhof-Rovers ◽  
JJ Sixma
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Divalent Cations ◽  
Platelet Adherence ◽  
Human Arteries ◽  
Platelet Spreading ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

The role of divalent cations in platelet adherence to deendothelialized human arteries in flowing blood was investigated in an annular perfusion chamber. Spreading of platelets on the subendothelium was impaired below 30 microM of free Ca2+ ions (Ca2+). When Ca2+ was replaced by Mg2+, adherence was unchanged in perfusates without exogenous factor VIII-von Willebrand factor (FVIII-vWF), but the ability of FVIII-vWF to support platelet adherence was lost. Binding of FVIII-vWF to the vessel wall was independent of divalent cations, but bound FVIII-vWF was only able to mediate adherence after exposure to Ca2+. Pretreatment of FVIII-vWF with the calcium chelator EGTA (10 mM) resulted in loss of the ability to facilitate platelet adherence, while the ristocetin cofactor activity remained intact. Full restoration of the ability to mediate platelet adherence could only be obtained by prolonged dialysis against Ca2+ in the millimolar range. These data indicate that divalent cations have at least two separate roles to play in supporting platelet adherence: (1) platelet spreading on the subendothelium requires Ca2+ or Mg2+; (2) FVIII-vWF should be exposed to Ca2+ to obtain its optimal biologic activity in supporting platelet adherence.

scholarly journals Immunologic studies of native and modified human factor VIII/von Willebrand factor

Blood ◽  
1979 ◽  
Vol 54 (2) ◽  
pp. 310-321 ◽  
Author(s):  
ME Switzer ◽  
PA McKee
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Procoagulant Activity ◽  
Human Factor Viii ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Factor VIII/von Willebrand factor (FVIII/vWF) is a glycoprotein with a molecular weight greater than one-million daltons. Two activities are associated with this large molecule: FVIII procoagulant activity and vWF activity. Incubation of FVIII/vWF with proteolytic enzymes causes rapid inactivation of the FVIII procoagulant activity but has little effect on the vWF activity or antigenicity. In an attempt to gain insight into the structural features required for these two activities, antisera were raised in rabbits to normal, thrombin-inactivated, and plasmin-inactivated FVIII/vWF. All of these proteolytically modified forms of FVIII/vWF cross-reacted with each of the rabbit antisera; each blocked the ability of a human inhibitor to inactivate native active FVIII/vWF. Each of the antisera was a potent inhibitor of vWF activity and inactivated vWF activity at the same titer. The antisera were much less potent inhibitors of FVIII activity than of vWF activity. Antibodies to thrombin-inactivated FVIII/vWF or normal FVIII/vWF had about the same ability to inactivate FVIII procoagulant activity. Surprisingly, those to plasmin-inactivated FVIII/vWF still retained about 50% of this inhibitory capacity. A comparison of the three types of antigens by polyacrylamide gel electrophoresis in sodium dodecyl sulfate-6 M urea demonstrated that the structure of thrombin- inactivated FVIII/vWF was indistinguishable from that of normal FVIII/vWF, while plasmin-inactivated FVII/vWF was completely cleaved to lower molecular weight fragments. Some of the reported variations in the ability of rabbit antibodies to inhibit procoagulant activity may be due to partial degradation of the starting antigen. The retention by FVIII/vWF protein of its immunologic properties even after extensive proteolytic degradation suggests that under nondenaturing conditions, the conformation of the native and degraded molecules are very similar.

scholarly journals Human platelet surface binding of endogenous secreted factor VIII-von Willebrand factor and platelet factor 4

Blood ◽  
1982 ◽  
Vol 59 (1) ◽  
pp. 194-197 ◽  
Author(s):  
JN George ◽  
AR Onofre
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Membrane Surface ◽  
Platelet Factor 4 ◽  
Secreted Proteins ◽  
Surface Binding ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Washed human platelets in buffers containing either 2 mM Ca++ or 4 mM EDTA were stimulated by human alpha-thrombin to induce secretion. The binding of two endogenous secreted proteins, factor-VIII-related protein (VIII-R) (von Willebrand factor) and platelet factor 4, was measured by reacting thrombin-treated and control platelets with specific antibodies to these proteins, then quantifying antibody binding with 125I-staphylococcal protein A. Both of these granule proteins were associated with the platelet membrane surface by a calcium-dependent mechanism after thrombin-induced secretion. This ability to bind endogenous secreted proteins to the plasma membrane surface may provide a mechanism by which the platelet can concentrate and organize its secreted proteins for subsequent physiologic reactions.

scholarly journals Immunologic studies of native and modified human factor VIII/von Willebrand factor

Blood ◽  
1979 ◽  
Vol 54 (2) ◽  
pp. 310-321
Author(s):  
ME Switzer ◽  
PA McKee
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Procoagulant Activity ◽  
Human Factor Viii ◽  
Von Willebrand ◽  
Willebrand Factor

Factor VIII/von Willebrand factor (FVIII/vWF) is a glycoprotein with a molecular weight greater than one-million daltons. Two activities are associated with this large molecule: FVIII procoagulant activity and vWF activity. Incubation of FVIII/vWF with proteolytic enzymes causes rapid inactivation of the FVIII procoagulant activity but has little effect on the vWF activity or antigenicity. In an attempt to gain insight into the structural features required for these two activities, antisera were raised in rabbits to normal, thrombin-inactivated, and plasmin-inactivated FVIII/vWF. All of these proteolytically modified forms of FVIII/vWF cross-reacted with each of the rabbit antisera; each blocked the ability of a human inhibitor to inactivate native active FVIII/vWF. Each of the antisera was a potent inhibitor of vWF activity and inactivated vWF activity at the same titer. The antisera were much less potent inhibitors of FVIII activity than of vWF activity. Antibodies to thrombin-inactivated FVIII/vWF or normal FVIII/vWF had about the same ability to inactivate FVIII procoagulant activity. Surprisingly, those to plasmin-inactivated FVIII/vWF still retained about 50% of this inhibitory capacity. A comparison of the three types of antigens by polyacrylamide gel electrophoresis in sodium dodecyl sulfate-6 M urea demonstrated that the structure of thrombin- inactivated FVIII/vWF was indistinguishable from that of normal FVIII/vWF, while plasmin-inactivated FVII/vWF was completely cleaved to lower molecular weight fragments. Some of the reported variations in the ability of rabbit antibodies to inhibit procoagulant activity may be due to partial degradation of the starting antigen. The retention by FVIII/vWF protein of its immunologic properties even after extensive proteolytic degradation suggests that under nondenaturing conditions, the conformation of the native and degraded molecules are very similar.

scholarly journals Human platelet surface binding of endogenous secreted factor VIII-von Willebrand factor and platelet factor 4

Blood ◽  
1982 ◽  
Vol 59 (1) ◽  
pp. 194-197 ◽  
Author(s):  
JN George ◽  
AR Onofre
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Membrane Surface ◽  
Platelet Factor 4 ◽  
Secreted Proteins ◽  
Surface Binding ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

Washed human platelets in buffers containing either 2 mM Ca++ or 4 mM EDTA were stimulated by human alpha-thrombin to induce secretion. The binding of two endogenous secreted proteins, factor-VIII-related protein (VIII-R) (von Willebrand factor) and platelet factor 4, was measured by reacting thrombin-treated and control platelets with specific antibodies to these proteins, then quantifying antibody binding with 125I-staphylococcal protein A. Both of these granule proteins were associated with the platelet membrane surface by a calcium-dependent mechanism after thrombin-induced secretion. This ability to bind endogenous secreted proteins to the plasma membrane surface may provide a mechanism by which the platelet can concentrate and organize its secreted proteins for subsequent physiologic reactions.

scholarly journals Subcellular platelet factor VIII antigen and von Willebrand factor.

1975 ◽  
Vol 141 (5) ◽  
pp. 1101-1113 ◽  
Author(s):  
R L Nachman ◽  
E A Jaffe
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Human Platelets ◽  
Factor Vii ◽  
Platelet Factor ◽  
Plasma Factor ◽  
Factor Viii Antigen ◽  
Von Willebrand ◽  
Willebrand Factor

Subcellular membrane and granule fractions derived from human platelets contain factor VIIII antigen and von Willebrand factor activity but not factor VII procoagulant activity. Circulating platelets constitute a significant reservoir of plasma factor VIII antigen, containing approximately 15% of the amount of factor VIII antigen present in platelet-poor plasma. The antibiotic ristocetin, which aggregates human platelets in the presence of von Willebrand factor, nonspecifically precipitates platelet membrane factor VIII antigen. Thus normal platelets contain surface-bound as well as internally stored von Willebrand factor, a protein synthesized by endothelial cells which is necessary for normal platelet function in vivo.

scholarly journals Factor VIII-von Willebrand factor requires calcium for facilitation of platelet adherence

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 996-103 ◽  
Author(s):  
KS Sakariassen ◽  
M Ottenhof-Rovers ◽  
JJ Sixma
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Divalent Cations ◽  
Platelet Adherence ◽  
Human Arteries ◽  
Platelet Spreading ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

Abstract The role of divalent cations in platelet adherence to deendothelialized human arteries in flowing blood was investigated in an annular perfusion chamber. Spreading of platelets on the subendothelium was impaired below 30 microM of free Ca2+ ions (Ca2+). When Ca2+ was replaced by Mg2+, adherence was unchanged in perfusates without exogenous factor VIII-von Willebrand factor (FVIII-vWF), but the ability of FVIII-vWF to support platelet adherence was lost. Binding of FVIII-vWF to the vessel wall was independent of divalent cations, but bound FVIII-vWF was only able to mediate adherence after exposure to Ca2+. Pretreatment of FVIII-vWF with the calcium chelator EGTA (10 mM) resulted in loss of the ability to facilitate platelet adherence, while the ristocetin cofactor activity remained intact. Full restoration of the ability to mediate platelet adherence could only be obtained by prolonged dialysis against Ca2+ in the millimolar range. These data indicate that divalent cations have at least two separate roles to play in supporting platelet adherence: (1) platelet spreading on the subendothelium requires Ca2+ or Mg2+; (2) FVIII-vWF should be exposed to Ca2+ to obtain its optimal biologic activity in supporting platelet adherence.

scholarly journals An autosomal dominant, qualitative platelet disorder associated with multimerin deficiency, abnormalities in platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen and an epinephrine aggregation defect

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 4967-4978 ◽  
Author(s):  
CP Hayward ◽  
GE Rivard ◽  
WH Kane ◽  
J Drouin ◽  
S Zheng ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor V ◽  
Binding Studies ◽  
Platelet Factor ◽  
Platelet Disorder ◽  
Plasma Factor ◽  
Von Willebrand ◽  
Willebrand Factor

Multimerin is a massive soluble, multimeric protein found in platelets and endothelial cells. Recent studies identified multimerin as a specific coagulation factor V binding protein, complexed with platelet, but not plasma, factor V. These findings led us to investigate individuals with inherited factor V deficiencies for possible multimerin abnormalities. Platelet proteins were evaluated using immunoassays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, immunoprecipitation, and direct binding studies. Patients with factor V Quebec, a disorder with abnormal platelet factor V, had a quantitative deficiency in multimerin (n = 11 tested; mean, 12.5%; range, 5% to 27% of the normal pool; normal range, 45% to 214%) with a normal multimer pattern. Quantitative and qualitative abnormalities were detected in their platelet factor V. An unrelated patient who was deficient in platelet and plasma factor V had normal platelet multimerin. The levels of platelet beta- thromboglobulin, von Willebrand factor, thrombospondin, and fibrinogen antigen were normal in the factor V Quebec patients. However, proteins with abnormal mobility were detected in their platelet lysate and releasate, and their platelet thrombospondin, von Willebrand factor, and fibrinogen showed evidence of proteolytic degradation. Platelet counts of the factor V Quebec patients ranged from mildly thrombocytopenic to low normal (mean, 159 x 10(9)/L; range, 104 to 198 x 10(9)/L). In addition, their platelets failed to aggregate in response to 6 to 10 micromol/L epinephrine despite normal numbers of platelet alpha 2-adrenergic receptors. These data indicate that patients with factor V Quebec have an inherited bleeding disorder distinct from other platelet disorders and associated with multiple abnormalities, including multimerin deficiency, abnormal platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen, and an epinephrine aggregation defect.

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